1. Field of the Invention
The present invention relates to a composition for detecting the undifferentiated human pluripotent stem cells comprising an agent useful for measuring the level of Desmoglein 2 (Dsg2) mRNA or the protein thereof, a kit for detecting the undifferentiated human pluripotent stem cells comprising the said composition, a method for detecting the undifferentiated human pluripotent stem cells containing the step of measuring the level of Desmoglein 2 mRNA or the protein thereof, a method for evaluating the differentiation of human pluripotent stem cells and thereafter for separating the undifferentiated human pluripotent stem cells, a method for reducing the undifferentiated status of human pluripotent stem cells by inhibiting the expression or activation of Desmoglein 2, and a monoclonal antibody conjugating specifically to human Desmoglein 2.
2. Description of the Related Art
Stem cell is the cell that has a potential for unlimited proliferation as remains undifferentiated status and is capable of being differentiated into a specific cell with a unique function and shape once certain environment and conditions are given. Human pluripotent stem cell is a self-renewal cell in a certain in vitro culture condition. Owing to its characteristics of being differentiated into almost every cell forming a living subject, it has been an important target of study not only to understand basic knowledge on the development, differentiation, and growth of a subject but also to develop an agent for cell therapy which is believed to be a fundamental treatment method for the damage or injury of a subject or for various diseases, to screen a various novel drug candidates and their medicinal effects, to disclose a cause of disease, and to develop a treatment method, etc.
One of the pluripotent stem cells, embryonic stem cell, unlike the differentiated cell arrested in the cell cycle, can produce the same cell as itself by cell division, which is called self-renewal. Embryonic stem cell displays pluripotency that is the ability to be differentiated into almost every functional cell in human body under a certain environment or stimulus. So, it is expected to induce the differentiation of the embryonic stem cell into a specific target cell when a specific cell or organ is damaged by accident or disease. Accordingly, the treatment method using the embryonic stem cell rises as a fundamental treatment method for various incurable diseases.
Human induced pluripotent stem cell (iPSC) is also one of those pluripotent stem cells, that induces pluripotency of the cell that has been finished with differentiation so as to make the cell to be re-differentiated again. This stem cell also has the self-renewal ability like embryonic stem cell, indicating that this stem cell can also be able to be differentiated into almost every kind of cell. According to the reports made so far, human induced pluripotent stem cell has similar characteristics in gene expression and differentiation capability to pluripotent embryonic stem cell (Takahashi, K et al., Cell, 131:861-872, 2007).
To obtain the cells differentiated from human pluripotent stem cells, human pluripotent stem cells are first induced to be differentiated into a specific type of cells; and then the differentiated cells are conjugated with surface markers for recognition; and then the recognized cells are separated by FACS (Fluorescent Activated Cells Sorter) (Fukuda, H et al., Stem Cells (2006) (24.3: 763-771)), or the differentiated cells are labeled with antibody and then the labeled cells are separated by MACS (Magnetic Activated Cell Sorting) (David, R et al., Stem Cells (2005) (23.4: 77-82)). MACS is known to outperform FACS since it can eliminate the risk of cell exposure on laser necessary for FACS.
In the method for obtaining the differentiated cells, either the cell separation is performed by FACS or by MACS, the possibility of mixed-existence with undifferentiated pluripotent stem cells is not completely excluded, suggesting that both methods are limited in separating the differentiated cells alone with 100% purity. That is, the differentiated cells originated from pluripotent stem cells might be mixed with the undifferentiated cells, and there might be a risk of the undifferentiated pluripotent stem cells to cause a tumor called teratoma, which has been a continuous issue in the development of cell therapy products. Therefore, it is requested to develop a novel method to eliminate selectively the undifferentiated cells having the risk of causing teratoma alone with leaving the differentiated cells.
Recently, Oct-4, Nanog and Sox-2, which are involved in self-renewal and pluripotency, have been used as intracellular markers for the separation of human pluripotent stem cells. TRA-1-60, TRA-1-81, SSEA3, and SSEA4 antibodies have been used as cell surface markers, however the most of molecules recognized by these antibodies have carbohydrate epitope or have the functions that are not necessary for self-renewal or pluripotency of human pluripotent stem cell (Badcock, et al., Cancer Res. 59:4715-4719, 1999; Kannagi et al., EMBO. J. 2:2355-2361, 1983; Brimble et al., Stem Cells. 25:54-62, 2007). Therefore, it is necessary to develop cell surface markers that are expressed in human pluripotent stem cells in order to study or separate the undifferentiated human pluripotent stem cells. Likewise, a novel agent that can recognize specifically human pluripotent stem cells and accordingly can be used efficiently to eliminate the undifferentiated human pluripotent stem cells during cell therapy is highly requested.
Under these circumstances, the present inventors tried to develop a novel method and technique to detect and separate specifically the undifferentiated human pluripotent stem cells. As a result, the inventors identified Desmoglein 2 which is specifically expressed in the undifferentiated human pluripotent stem cells and thereafter prepared an agent that can be conjugated specifically to the undifferentiated pluripotent stem cells, and further confirmed that the detection and separation of the undifferentiated human pluripotent stem cells could be achieved by this method by using the agent, leading to the completion of this invention.